The present invention relates to the isolation of proteins from animal cells, particularly mammalian cells, that bind to human rhinovirus (HRV). More particularly, the invention relates to the isolation of HRV receptor proteins that can bind to HRV and thereby block the infectivity of the virus. This property can serve as a basis for inhibiting the initiation or the spread of HRV infections, better known as the common cold.
In order to infect host cells, viruses must bind to and then enter cells to initiate an infection. Since 1959, evidence has accumulated in the literature indicating that the presence of specific binding sites (receptors) on host cells could be a major determinant of tissue tropism of certain viruses. [Holland, J. J., and McLaren, L. C., The mammalian cell-virus relationship, II. Absorption, reception, and eclipse of poliovirus by HeLa cells, J. Exp. Med. 109, 487-504 (1959). Holland, J. J., Receptor affinities as major determinants of enterovirus tissue tropisms in humans, Virology 15, 312-326 (1961).] Among picornaviruses such as poliovirus, coxsacchie virus, and rhinoviruses, specific binding to host cells has been demonstrated. By competition experiments, it has been demonstrated that some of these receptors are distinct from one another in that the saturation of the receptor of one virus had no effect on the binding of a second virus. [Lonberg-Holm, K, Crowell, R. L., and Philipson, L. Unrelated animal viruses share receptors, Nature 259, 679-681 (1976)].
Rhinoviruses form the largest family of picornaviruses, with 115 distinct serotypes identified to date. A large fraction of rhinoviruses (estimated to be 80%) appear to bind to a common receptor on human cells. [Abraham, G., and Colonno, R. J., Many rhinovirus serotypes share the same cellular receptor, J. of Virology 51, 340-345 (1984).] In 1985, the isolation of a monoclonal antibody that appeared to be directed against the major rhinovirus receptor was described. [Colonno, R. J., Callahan, P. L., and Long, W. J., Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses, J. of Virology 57, 7-12 (1986).] It inhibited infection of cells with the appropriate serotypes of rhinovirus and it inhibited binding of radiolabeled rhinovirus to cells. This group subsequently reported that the monoclonal antibody bound to a protein with an apparent molecular weight of 90,000 daltons. Tomassini, J. E., and Colonno, R. J., Isolation of a receptor protein involved in attachment of human rhinoviruses, J. of Virology 58, 290-295 (1986). This monoclonal antibody has been utilized in clinical trials with primates and humans and is understood to provide some protection against rhinovirus infection.
There are several other reports of attempts at therapeutic intervention in rhinovirus infections. Intranasal application of interferon in humans has been attempted. [Douglas, R. M., et al., Prophylactic efficacy of intranasal alpha2-interferon against rhinovirus infections in the family setting, The New England J. of Medicine, 314, 65-75 (1986).] In this case, significant reduction in the severity of the infection was found, although nosebleeds were observed as a side-effect. Also, several analogs of disoxaril ("WIN" compounds) that reduce the infectivity of a number of picornaviruses (with widely varying effectiveness, depending on the serotype) have been tested in tissue culture and in some animal models. [Fox, M. P., Otto, M. J., and McKinlay, M. A., Antimicrob. Ag. and Chemotherapy 30, 110-116 (1986).] These compounds appear to inhibit replication at a step subsequent to receptor binding, probably at some step of virus uncoating. The atomic coordinates of the binding sites of these compounds within the viral capsid of the serotype HRV14 have been determined by x-ray crystallography, and are located in a hydrophobic pocket present in each protomeric unit of the capsid. [Smith, T. J., et al., The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating, Science 233, 1286-1293 (1986).] The specific function of the binding pocket, if any, is unknown, but drug-resistant mutants with single amino acid interchanges in this region arise at high frequency and are viable. [Badger, J. et al., Structural analysis of a series of antiviral agents complexed with human rhinovirus 14, PNAS 85, 3304-3308 (1988).] This result calls into question the efficacy of such compounds as drugs. The production of anti-peptide antibodies in rabbits has been reported using peptides derived from amino acid sequence of the viral capsid proteins that line the "receptor canyon" of HRV14. [McCray, J., and Werner, G., Different rhinovirus serotypes neutralized by antipeptide antibodies, Nature 329:736-738 (1987).] While the titers of these sera are quite low, cross-serotype protection of cells in tissue culture from rhinovirus infection was demonstrated, raising the possibility of a vaccine.
It is an object of the present invention to isolate an HRV receptor protein from cells having the property of blocking HRV infection. Given the high affinity the virus has for its receptor, it was hypothesized that a therapeutic agent effective against HRV infection might be the receptor itself, or more specifically, the virus binding domain of the receptor. A protein, protein fragment, or peptide that comprises the virus binding domain could block the ability of virus to bind to host cells by occupying (blocking) the receptor binding cleft on the virus. Furthermore, since such a molecule would make some or all of the molecular contacts with the virus capsid that the receptor does, virus mutations that adversely affect binding of the molecule would adversely affect binding of the receptor, and would thus be deleterious or lethal for the virus; therefore, the likelihood of drug-resistant mutants would be very low. Furthermore, such a molecule would be human, lowering the likelihood of being antigenic in humans.